R-3 Repository :: Browsing by Author "Warmflash, Aryeh"

Browsing by Author "Warmflash, Aryeh"

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A novel self-organizing embryonic stem cell system reveals signaling logic underlying the patterning of human ectoderm in two- and three-dimensions
(2020-08-04) Britton, George Leslie; Warmflash, Aryeh
During development, the ectoderm is patterned by a combination of BMP and WNT signaling. Research in model organisms has provided substantial insight into this process; however, there are currently no systems in which to study ectodermal patterning in humans as it transitions from a two-dimensional to a three-dimensional tissue. Further, the complexity of neural plate border specification has made it difficult to transition from discovering the genes involved to deeper mechanistic understanding. Here, we develop an in vitro model of human ectodermal patterning in both two- and three-dimensions, in which human embryonic stem cells self-organize to form robust and quantitatively reproducible patterns corresponding to the complete medial-lateral axis of the embryonic ectoderm. Importantly, this organized tissue undergoes morphogenesis to form a close neural tube with an overlaying surface ectoderm, similar to what is found in vivo. Using this platform, we show that the duration of endogenous WNT signaling is a crucial control parameter, and that cells sense relative levels of BMP and WNT signaling in making fate decisions. These insights allowed us to develop an improved protocol for placodal differentiation in standard culture. Thus, our platform is a powerful tool for studying human ectoderm patterning and three-dimensional self-organization of neural structures that is otherwise impossible to study in utero.
Barcoding tools to track development and function of stem cell constructs for biomedicine
(2019-04-04) Mitra, Kinshuk; Warmflash, Aryeh
Biological regeneration has long been explored as a breakthrough modality for human therapy. Degenerative diseases, a majority of which are a related to ageing, manifest as a wide variety of pathologies: cardiovascular diseases, diabetes and Alzheimer to name a few. Consequently, the idea of a panacea in the form of a biological therapeutic that can grow and restore a debilitated bodily function has been much investigated. Outwardly, the premise has seemed remarkably simple. Scientists have long observed lizards growing back whole tails after decapitation and studied the remarkable regeneration potential of Planaria. Yet increasing work suggests processes including complex cellular growth dynamics, patterns of gene expression and clonal expansion of cells drives regeneration and analogously, biological tissue development. Improved understanding of developmental biology can thus improve the function and persistence of regenerative therapies. Our lab has previously developed in-vitro tools that allow the study of biological development. This work extends on that body by developing two distinct tool sets. Firstly, we develop a scheme for tracing large-scale clonal dynamics conveniently using a scalable lineage tracing method. We demonstrate the capacity of this system to accurately detect cell lineages based on a system of inheritable genetic barcoding. Our system can deconvolute lineage mixtures, track growth dynamics and consequently probe biological phenomena. Importantly, our process preserves spatial integrity of the sample and thus quantifies both lineage dynamics and captures their positional information. Since differential patterns of gene expression is another important component of regeneration and development, we develop a method to measure gene expression non-destructively, with the capacity to allow multiplexed monitoring of multiple targets even while in-vivo. As cellular and regenerative therapies move through primate and clinical trials, such a tool can serve the critical role of augmenting available biomarkers for monitoring therapy and predicting clinical outcomes thereby improving regulatory clearance. Taken together, our work adds to the body of knowledge that seeks to better understand tissue genesis and its re-engineering to create new therapeutic modalities.
BMP-treated human embryonic stem cells transcriptionally resemble amnion cells in the monkey embryo
(The Company of Biologists, 2021) Chhabra, Sapna; Warmflash, Aryeh
Human embryonic stem cells (hESCs) possess an immense potential to generate clinically relevant cell types and unveil mechanisms underlying early human development. However, using hESCs for discovery or translation requires accurately identifying differentiated cell types through comparison with their in vivo counterparts. Here, we set out to determine the identity of much debated BMP-treated hESCs by comparing their transcriptome to recently published single cell transcriptomic data from early human embryos ( Xiang et al., 2020). Our analyses reveal several discrepancies in the published human embryo dataset, including misclassification of putative amnion, intermediate and inner cell mass cells. These misclassifications primarily resulted from similarities in pseudogene expression, highlighting the need to carefully consider gene lists when making comparisons between cell types. In the absence of a relevant human dataset, we utilized the recently published single cell transcriptome of the early post implantation monkey embryo to discern the identity of BMP-treated hESCs. Our results suggest that BMP-treated hESCs are transcriptionally more similar to amnion cells than trophectoderm cells in the monkey embryo. Together with prior studies, this result indicates that hESCs possess a unique ability to form mature trophectoderm subtypes via an amnion-like transcriptional state.This article has an associated First Person interview with the first author of the paper.
Characterizing the Host-Pathogen Interactions and Inter-Strain Competition in Pathogenic Bacteria
(2023-06-01) Zhang, Liyang; Kirienko, Natasha V; Warmflash, Aryeh; Gao, Yang; van der Hoeven, Ransome V; Igoshin, Oleg A
Pathogens are of significant importance in medicine and public health, as they are responsible for a wide range of infectious diseases that can have serious consequences for human and animal populations. Whether infection gets established, depends on multiple factors, including environment, the host, and interactions with other bacteria. Understanding these factors and their impact is a prerequisite for developing therapies for resisting or disarming pathogenic bacteria. Adhesins are proteins present on the microbial cell surface that mediate the interactions with or attachment to the host or substance. As one type of virulence factors, adhesin proteins play a crucial role in the ability of the fungal pathogen Candida albicans to undergo cellular morphogenesis, develop robust biofilms, colonize, and cause infection in a host. By performing a comprehensive, high-throughput screen of a library of adhesin mutants in the model nematode Caenorhabditis elegans as a simplified host system, I identified mutants critical for virulence of C. albicans. Colonization is generally considered a prerequisite for infection, but this event is context-dependent, as evidenced by the differing ability of the human pathogen Pseudomonas aeruginosa to efficiently colonize C. elegans on agar but not in liquid pathogenesis. I showed that the transition to a liquid environment reduces food uptake, decreases specific adhesins, slightly upregulates host immunity, and induces a pathogen-driven dormancy of C. elegans, which restricts pathogenic colonization. My study also found that pathogenic colonization was still required for the virulence of Enterococcus faecalis even in the liquid. I conclude that poor colonization in liquid is likely due to a combination of environmental factors and host-pathogen interactions. These results provide new insights into mechanisms for colonization in different models, enabling pathogenesis models to be fine-tuned to more accurately represent the conditions seen in human infections so that new tools for curbing bacterial and fungal infections can be developed. Competition shapes the life spectrum in nature, resulting in organisms with better fitness taking a position of dominance and prevalence. The high-risk clone of P. aeruginosa ST111 predominates in hematopoietic cell transplant and hematologic malignancy (HCT/HM) bloodstream infection (BSI) patients via a fitness benefit due to the loss of functional OprD, a porin responsible for the import of carbapenems. Further study revealed that not only ST111 but also several international high-risk sequence types produce the bactericidal R5 pyocin that targets P. aeruginosa with mutations on WaaL, an O-antigen ligase of lipopolysaccharide. These findings suggest a novel approach for evaluating risks associated with emerging prevalent P. aeruginosa strains and may inform the development of strategies to mitigate the impact of ST111 and other high-risk clones on public health. In conclusion, my dissertation research provides valuable insights into the virulence of pathogens and their interactions with the host. Having a thorough understanding of the features of each infection model can power researchers to do pathogenesis research and hereby develop effective treatments or interventions for infectious diseases. The identification of R5 pyocin producers and their fitness benefits in causing infection highlights the need for ongoing monitoring and surveillance to inform public health strategies to mitigate the impact of emerging pathogens on human health.
Coco is a dual activity modulator of TGFβ signaling
(The Company of Biologists, 2015) Deglincerti, Alessia; Haremaki, Tomomi; Warmflash, Aryeh; Sorre, Benoit; Brivanlou, Ali H.
The TGFβ signaling pathway is a crucial regulator of developmental processes and disease. The activity of TGFβ ligands is modulated by various families of soluble inhibitors that interfere with the interactions between ligands and receptors. In an unbiased, genome-wide RNAi screen to identify genes involved in ligand-dependent signaling, we unexpectedly identified the BMP/Activin/Nodal inhibitor Coco as an enhancer of TGFβ1 signaling. Coco synergizes with TGFβ1 in both cell culture and Xenopus explants. Molecularly, Coco binds to TGFβ1 and enhances TGFβ1 binding to its receptor Alk5. Thus, Coco acts as both an inhibitor and an enhancer of signaling depending on the ligand it binds. This finding raises the need for a global reconsideration of the molecular mechanisms regulating TGFβ signaling.
Dissecting signaling dynamics underlying self-organized fate patterning of 2D human gastruloids
(2020-04-13) Chhabra, Sapna; Warmflash, Aryeh
Gastrulation is a crucial stage in embryonic development when the epiblast self- organizes to form three germ layers – the ectoderm, mesoderm and endoderm, which eventually form the entire organism. Decades of genetic and biochemical studies in the mouse embryo have revealed that a signaling cascade involving the BMP, WNT and NODAL pathways is necessary for gastrulation. In the developing mouse embryo, WNT and NODAL ligands are expressed near the site of gastrulation, and knockout of these ligands leads to a failure to gastrulate. These data have led to the prevailing view that a signaling gradient in WNT and NODAL underlies germ layer fate patterning during gastrulation. However, the activities of these pathways in space and time have never been directly observed, as gastrulation occurs after the mouse embryo implants in the mother’s uterus, and thus, precludes, direct observation and analyses. Thus, whether a stable signaling gradient of WNT and NODAL activities underlies formation of the three germ layers remains unknown. In this study, we utilize our previously developed model of human gastrulation (2D human gastruloids) where circular colonies of human embryonic stem cells (hESCs), treated with BMP4 ligands, self-organize to form a radial pattern of the three germ layers surrounded by extra-embryonic cells at the colony edge; to measure signaling activities of WNT and NODAL. Our data shows that BMP signaling initiates a wave of WNT signaling that initiates a wave of NODAL signaling. Both WNT and NODAL signaling activities spread towards the colony center at a constant rate. Using a simple mathematical model, we show that this spreading behavior is inconsistent with a reaction- diffusion–based Turing system, indicating that there is no stable signaling gradient of WNT/NODAL signaling activity. Instead, the final signaling state is homogeneous, and spatial differences arise only from boundary effects. We further show that although both WNT and NODAL signaling synergize to enable mesodermal differentiation, neither of them forms a spatial pattern that maps directly to the mesodermal region, suggesting that mesoderm differentiation is controlled by the dynamics of multiple signals. In contrast to this, extra-embryonic differentiation at the colony edge is controlled by the duration of BMP signaling, which is consistently high in extra-embryonic cells. The identity of these extra-embryonic cells has been controversial, with some studies suggesting a trophoblast cell fate and others suggesting an extra-embryonic mesodermal fate. Using RNA- sequencing, we show that these cells are transcriptionally similar to trophoblast cells in the day 7 human embryo. Taken together, our results show that that the dynamics of signaling events in the BMP, WNT, and NODAL cascade in the absence of a stable signaling gradient, instruct germ layer fate patterning in human gastruloids.
Dissecting the dynamics of signaling events in the BMP, WNT, and NODAL cascade during self-organized fate patterning in human gastruloids
(Public Library of Science, 2019) Chhabra, Sapna; Liu, Lizhong; Goh, Ryan; Kong, Xiangyu; Warmflash, Aryeh
microfiber mesh function to maintain scaffold cellularity under serum-free conditions as well as aid the deposition of GAGs. This supports the hypothesis that scaffolds with constituents more closely resembling native ECM components may be beneficial for cartilage regeneration.
Endogenous Nodal diverges Wnt signaling interpretation from definitive endoderm to posterior mesoderm in geometrically constrained human pluripotent cells.
(2023-11-28) Ortiz Salazar, Miguel Ángel; Stern, Michael; Warmflash, Aryeh
Embryonic development is a dynamic and highly complex process. It demands the intricate coordination of several mechanisms and signaling pathways to regulate cell fate determination, tissue patterning, and functional integrity, ensuring the embryo’s proper formation until birth. One crucial signaling cascade in development is the Wnt pathway. It is essential during the early stages, as it directs gastrulation, the phase in which the embryo starts forming the germ layers, particularly the endoderm and mesoderm. The Wnt pathway also plays a role in establishing the anterior-to-posterior body axis and helps in elongating the body. However, the precise mechanisms of how Wnt coordinates these diverse stages yielding different outcomes remain not fully elucidated. In this work, we aimed to study the Wnt pathway and its role in cell fate decisions. We uncovered that a posterior-progenitor-like state is induced when the posterior signals, WNT3a and FGF8 are presented to human embryonic stem cell colonies in standard culture. However, when these colonies are geometrically constrained, colonies self- organize themselves into a structure composed of a definitive endoderm cell population surrounding and lifting an epiblast disk-like structure. These unprecedented colonies do not change cell fate when Wnt levels are increased, challenging the classic concentration- dependent morphogen mechanism. While studying this discrepancy, we were able to determine that TGF-ß pathway is responsible for maintaining the definitive endoderm cell fates. Only when this pathway was perturbed, cells were able to respond in a concentration-dependent manner to Wnt and acquire posterior mesoderm cell fates. Upon further inquiry, we were able to determine that specifically, Nodal is the main driver for endoderm cell fates. Finally, we determined that CHIR, a commonly used chemical Wnt activator, induces posterior cell fates in a concentration-dependent manner qualitatively different from induced WNT signaling dynamics and Nodal ligand production. Collectively, we demonstrate that cell fate decision-making is determined by the interplay between multiple pathways and not by the levels of a single pathway, highlighting the dynamic nature of development.
Endothelial cells decode VEGF-mediated Ca2+ signaling patterns to produce distinct functional responses
(American Association for the Advancement of Science, 2016) Noren, David P.; Chou, Wesley H.; Lee, Sung Hoon; Qutub, Amina A.; Warmflash, Aryeh; Wagner, Daniel S.; Popel, Aleksander S.; Levchenko, Andre
A single extracellular stimulus can promote diverse behaviors among isogenic cells by differentially regulated signaling networks. We examined Ca2+ signaling in response to VEGF (vascular endothelial growth factor), a growth factor that can stimulate different behaviors in endothelial cells. We found that altering the amount of VEGF signaling in endothelial cells by stimulating them with different VEGF concentrations triggered distinct and mutually exclusive dynamic Ca2+ signaling responses that correlated with different cellular behaviors. These behaviors were cell proliferation involving the transcription factor NFAT (nuclear factor of activated T cells) and cell migration involving MLCK (myosin light chain kinase). Further analysis suggested that this signal decoding was robust to the noisy nature of the signal input. Using probabilistic modeling, we captured both the stochastic and deterministic aspects of Ca2+ signal decoding and accurately predicted cell responses in VEGF gradients, which we used to simulate different amounts of VEGF signaling. Ca2+ signaling patterns associated with proliferation and migration were detected during angiogenesis in developing zebrafish.
Membrane potential drives the exit from pluripotency and cell fate commitment via calcium and mTOR
(Springer Nature, 2022) Sempou, Emily; Kostiuk, Valentyna; Zhu, Jie; Cecilia Guerra, M.; Tyan, Leonid; Hwang, Woong; Camacho-Aguilar, Elena; Caplan, Michael J.; Zenisek, David; Warmflash, Aryeh; Owens, Nick D. L.; Khokha, Mustafa K.
Transitioning from pluripotency to differentiated cell fates is fundamental to both embryonic development and adult tissue homeostasis. Improving our understanding of this transition would facilitate our ability to manipulate pluripotent cells into tissues for therapeutic use. Here, we show that membrane voltage (Vm) regulates the exit from pluripotency and the onset of germ layer differentiation in the embryo, a process that affects both gastrulation and left-right patterning. By examining candidate genes of congenital heart disease and heterotaxy, we identify KCNH6, a member of the ether-a-go-go class of potassium channels that hyperpolarizes the Vm and thus limits the activation of voltage gated calcium channels, lowering intracellular calcium. In pluripotent embryonic cells, depletion of kcnh6 leads to membrane depolarization, elevation of intracellular calcium levels, and the maintenance of a pluripotent state at the expense of differentiation into ectodermal and myogenic lineages. Using high-resolution temporal transcriptome analysis, we identify the gene regulatory networks downstream of membrane depolarization and calcium signaling and discover that inhibition of the mTOR pathway transitions the pluripotent cell to a differentiated fate. By manipulating Vm using a suite of tools, we establish a bioelectric pathway that regulates pluripotency in vertebrates, including human embryonic stem cells.
Modeling human gastrulation at the extraemrbyonic-embryonic border
(2024-04-19) Kong, Xiangyu; Warmflash, Aryeh
Human gastrulation has long been a fascinating process that developmental biology tries to understand. Due to a lack of sample accessibility and ethical concerns, the answers to some basic questions like what signaling is initiating this process still elude us. In the past decade, several in vitro models were developed to answer such questions using human pluripotent stem cells (hPSCs), such as 2D and 3D human gastruloids. We gained a lot of insight into fate patterning, morphogenesis, and the mechanism driving them by studying these in vitro models. Compared to in vivo studies, these in vitro models provide us with better flexibility for experimental manipulation and scalability. However, the current in vitro models for human gastrulation are initiated by adding exogenous signaling molecules, by sticking to these systems, we risk missing some of the detailed dynamics during the process. It has long been hypothesized that the fate patterning during gastrulation is induced by signaling molecules secreted from the neighboring extraembryonic tissues, however, such inducing potency has yet to be reliably demonstrated in a human model. We developed a simple experimental model that juxtaposes extraembryonic and embryonic cells side by side to recreate a boundary to directly observe the interactions between them. We found that when hPSCs are juxtaposed against amnion-like cells (AMLCs), cells near the border recapitulating several aspects of gastrulation including…. We were able to capture this patterned differentiation and the underlying signaling in time. Studying the connection between said fate patterning and underlying signals grants us further insights into human gastrulation that have not been revealed in previous studies conducted on other model organisms or alternative gastrulation models. This demonstrates the potential of the juxtaposition model for studying the native signaling events between different cell populations.
Nodal is a short-range morphogen with activity that spreads through a relay mechanism in human gastruloids
(Springer Nature, 2022) Liu, Lizhong; Nemashkalo, Anastasiia; Rezende, Luisa; Jung, Ji Yoon; Chhabra, Sapna; Guerra, M. Cecilia; Heemskerk, Idse; Warmflash, Aryeh
Morphogens are signaling molecules that convey positional information and dictate cell fates during development. Although ectopic expression in model organisms suggests that morphogen gradients form through diffusion, little is known about how morphogen gradients are created and interpreted during mammalian embryogenesis due to the combined difficulties of measuring endogenous morphogen levels and observing development in utero. Here we take advantage of a human gastruloid model to visualize endogenous Nodal protein in living cells, during specification of germ layers. We show that Nodal is extremely short range so that Nodal protein is limited to the immediate neighborhood of source cells. Nodal activity spreads through a relay mechanism in which Nodal production induces neighboring cells to transcribe Nodal. We further show that the Nodal inhibitor Lefty, while biochemically capable of long-range diffusion, also acts locally to control the timing of Nodal spread and therefore of mesoderm differentiation during patterning. Our study establishes a paradigm for tissue patterning by an activator-inhibitor pair.
Quantifying cell transitions in C. elegans with data-fitted landscape models
(Public Library of Science, 2021) Camacho-Aguilar, Elena; Warmflash, Aryeh; Rand, David A.
Increasing interest has emerged in new mathematical approaches that simplify the study of complex differentiation processes by formalizing Waddington’s landscape metaphor. However, a rational method to build these landscape models remains an open problem. Here we study vulval development in C. elegans by developing a framework based on Catastrophe Theory (CT) and approximate Bayesian computation (ABC) to build data-fitted landscape models. We first identify the candidate qualitative landscapes, and then use CT to build the simplest model consistent with the data, which we quantitatively fit using ABC. The resulting model suggests that the underlying mechanism is a quantifiable two-step decision controlled by EGF and Notch-Delta signals, where a non-vulval/vulval decision is followed by a bistable transition to the two vulval states. This new model fits a broad set of data and makes several novel predictions.
Rapid changes in morphogen concentration control self-organized patterning in human embryonic stem cells
(eLife Sciences Publications, Ltd, 2019) Heemskerk, Idse; Burt, Kari; Miller, Matthew; Chhabra, Sapna; Guerra, M.Cecilia; Liu, Lizhong; Warmflash, Aryeh
During embryonic development, diffusible signaling molecules called morphogens are thought to determine cell fates in a concentration-dependent way. Yet, in mammalian embryos, concentrations change rapidly compared to the time for making cell fate decisions. Here, we use human embryonic stem cells (hESCs) to address how changing morphogen levels influence differentiation, focusing on how BMP4 and Nodal signaling govern the cell-fate decisions associated with gastrulation. We show that BMP4 response is concentration dependent, but that expression of many Nodal targets depends on rate of concentration change. Moreover, in a self-organized stem cell model for human gastrulation, expression of these genes follows rapid changes in endogenous Nodal signaling. Our study shows a striking contrast between the specific ways ligand dynamics are interpreted by two closely related signaling pathways, highlighting both the subtlety and importance of morphogen dynamics for understanding mammalian embryogenesis and designing optimized protocols for directed stem cell differentiation.
Rapid fabrication of hydrogel micropatterns by projection stereolithography for studying self-organized developmental patterning
(Public Library of Science, 2021) Zhu, Ye; Sazer, Daniel; Miller, Jordan S.; Warmflash, Aryeh
Self-organized patterning of mammalian embryonic stem cells on micropatterned surfaces has previously been established as an in vitro platform for early mammalian developmental studies, complimentary to in vivo studies. Traditional micropatterning methods, such as micro-contact printing (μCP), involve relatively complicated fabrication procedures, which restricts widespread adoption by biologists. Here, we demonstrate a rapid method of micropatterning by printing hydrogel micro-features onto a glass-bottomed culture vessel. The micro-features are printed using a projection stereolithography bioprinter yielding hydrogel structures that geometrically restrict the attachment of cells or proteins. Compared to traditional and physical photomasks, a digitally tunable virtual photomask is used in the projector to generate blue light patterns that enable rapid iteration with minimal cost and effort. We show that a protocol that makes use of this method together with LN521 coating, an extracellular matrix coating, creates a surface suitable for human embryonic stem cell (hESC) attachment and growth with minimal non-specific adhesion. We further demonstrate that self-patterning of hESCs following previously published gastrulation and ectodermal induction protocols achieves results comparable with those obtained with commercially available plates.
Roadmap for the multiscale coupling of biochemical and mechanical signals during development
(IOP Publishing, 2021) Lenne, Pierre-François; Munro, Edwin; Heemskerk, Idse; Warmflash, Aryeh; Bocanegra-Moreno, Laura; Kishi, Kasumi; Kicheva, Anna; Long, Yuchen; Fruleux, Antoine; Boudaoud, Arezki; Saunders, Timothy E.; Caldarelli, Paolo; Michaut, Arthur; Gros, Jerome; Maroudas-Sacks, Yonit; Keren, Kinneret; Hannezo, Edouard; Gartner, Zev J.; Stormo, Benjamin; Gladfelter, Amy; Rodrigues, Alan; Shyer, Amy; Minc, Nicolas; Maître, Jean-Léon; Talia, Stefano Di; Khamaisi, Bassma; Sprinzak, David; Tlili, Sham
The way in which interactions between mechanics and biochemistry lead to the emergence of complex cell and tissue organization is an old question that has recently attracted renewed interest from biologists, physicists, mathematicians and computer scientists. Rapid advances in optical physics, microscopy and computational image analysis have greatly enhanced our ability to observe and quantify spatiotemporal patterns of signalling, force generation, deformation, and flow in living cells and tissues. Powerful new tools for genetic, biophysical and optogenetic manipulation are allowing us to perturb the underlying machinery that generates these patterns in increasingly sophisticated ways. Rapid advances in theory and computing have made it possible to construct predictive models that describe how cell and tissue organization and dynamics emerge from the local coupling of biochemistry and mechanics. Together, these advances have opened up a wealth of new opportunities to explore how mechanochemical patterning shapes organismal development. In this roadmap, we present a series of forward-looking case studies on mechanochemical patterning in development, written by scientists working at the interface between the physical and biological sciences, and covering a wide range of spatial and temporal scales, organisms, and modes of development. Together, these contributions highlight the many ways in which the dynamic coupling of mechanics and biochemistry shapes biological dynamics: from mechanoenzymes that sense force to tune their activity and motor output, to collectives of cells in tissues that flow and redistribute biochemical signals during development.
Role of SULF1 in the regulation of metastatic prostate cancer progression
(2020-04-24) Brasil da Costa, Fabio Henrique; Carson, Daniel D; Warmflash, Aryeh
The establishment of new prostate cancer (PCa) metastases in the bone marrow accompanies a classical stromal reaction orchestrated primarily by the bidirectional engagement of cancer cells and fibroblasts. As result, tumor-associated macrophages (TAMs) are recruited to the tumor and display a fluid spectrum of behaviors that may promote or suppress tumor progression. These stromal and immune reactions, although initially in place to constrain tumor growth, have been shown to ultimately benefit cancer cells, for example, due to increased bioavailability of growth factors and matrix remodeling. Also, deposition of the heparan sulfate (HS) proteoglycan perlecan (HSPG2) and secretion of HS-modifying enzymes are increased in the matrix as result of the Cancer-CAF-TAM trialogue. In this work, to investigate PCa bone metastases, we developed a novel, bone marrow mimetic hydrogel containing collagen type I, hyaluronic acid, and HS-decorated domain 1 of perlecan (COL1-HA-PlnDm1). Our goal was to use this model to study the prevalence and role of growth factor-releasing enzymes, like sulfatases 1 and 2 (SULF1/SULF2) and heparanase. We first demonstrated that PCa cells, bone marrow fibroblasts, and Mφs have excellent viability, show in vivo-like morphology, and exhibit classical phenotypic markers in COL1-HA-PlnDm1 hydrogels. Furthermore, we found that SULF1 is the major HS modifier and is predominantly expressed by activated fibroblasts both in vivo and in vitro. Then, we discovered that TAMs are highly prevalent in PCa bone marrow metastases, as indicated by CD163 and CD206 expression, and Mφs in 3D displayed similar phenotype. Additionally, alternatively activated macrophages induced upregulation of SULF1 and HSPG2 in fibroblasts in vitro. Finally, our 3D triculture data shows that when SULF1 is knocked out from fibroblasts, PCa cell proliferation and cluster volume are significantly increased in response to treatment with Wnt3a, even in the presence of Mφs. We conclude that 1) our 3D hydrogel model is a physiologically relevant in vitro system to discover fundamental aspects of HS signaling in the tumor microenvironment; and 2) the dynamics of SULF1 expression and activity suggest a tumor suppressing role in the context of Wnt3a signaling in PCa. Further studies are necessary to determine SULF1’s value as a prognostic marker or a candidate target for new therapies.
Self-organized signaling in stem cell models of embryos
(Elsevier, 2021) Liu, Lizhong; Warmflash, Aryeh
Mammalian embryonic development is a complex process driven by self-organization. Understanding how a fertilized egg develops into an embryo composed of more than 200 cell types in precise spatial patterns remains one of the fundamental challenges in biology. Pluripotent stem cells have been used as in vitro models for investigating mammalian development, and represent promising building blocks for regenerative therapies. Recently, sophisticated stem cell-based models that recapitulate early embryonic fate patterning and morphogenesis have been developed. In this article, we review recent advances in stem cell models of embryos in particular focusing on signaling activities underpinning cell fate decisions in space and time.
Stem cell-based models of embryos: The need for improved naming conventions
(Elsevier, 2021) Matthews, Kirstin R.W.; Wagner, Daniel S.; Warmflash, Aryeh; James A. Baker III Institute for Public Policy-Center for Health and Biosciences
Stem cell-based models of embryos are known by various names, with different naming conventions, leading to confusion regarding their composition and potential. We propose the need for a general term for the field to promote public engagement and the development of a systematic nomenclature system to differentiate between specific models.
Transcriptional profiles of differentiating periocular neural crest cells and the function of Nephronectin during chick corneal development
(2020-04-22) Bi, Lian; Lwigale, Peter Y.; Warmflash, Aryeh
During eye formation, periocular neural crest cells (pNC) migrate and differentiate to form the anterior ocular structures. In the chick cornea, this involves two waves of migration that result in the formation of the corneal endothelium and stroma. Abnormalities in pNC migration lead to corneal malformation, such as anterior segment dystrophy. Corneal dystrophies, infections, and injuries can lead to corneal blindness, one of the major causes of blindness. Alternative treatments are developed because of the limitation of traditional corneal transplantation. These treatments benefit from the study of the molecular basis of corneal development and regeneration. However, corneal development is not fully understood. The purpose of this work was to elucidate the gene expression profiles during pNC migration and to examine the function of a highly regulated gene, Nephronectin (NPNT), during corneal formation. By performing RNA-seq analysis comparing pNC to its derived corneal structures, I analyzed differentially expressed genes and examined differentiated pathways during corneal formation. This project summarized the transcriptional regulation that happens at three levels: signaling pathways, transcription factors, and the downstream endothelial and stromal genes, providing gene candidates involved in corneal formation for future studies. From the RNA-seq analysis, I identified novel upregulation of NPNT among the extracellular matrix (ECM) proteins of the cornea. NPNT has been studied in other developmental processes but has not been linked to the corneal formation. Here, I found that NPNT is distributed in the primary stroma during pNC migration. Its receptor, Integrin (ITG) α8, is expressed in the pNC that migrate on the primary stroma. Thus, I hypothesized that NPNT interacts with ITGα8β1 to promote pNC migration during corneal development. I performed functional studies by the RCAS-RNAi system. The knockdown of either NPNT or ITGα8 resulted in the reduction of corneal stromal thickness. Further studies revealed that NPNT overexpression upregulated cell numbers in the corneal stroma but did not increase cell proliferation. Inhibition of ITGα8 in vivo and in vitro both reduced pNC migration. Together, the functional studies link NPNT/ITGα8β1 signaling to pNC migration during corneal formation. This study reveals a previously unknown ECM-receptor pathway in corneal formation, suggesting a potential gene target or culture matrix in corneal development and regeneration. Collectively, this work depicts the transcription profiles during chick embryonic corneal development and investigates the function of a candidate ECM protein, NPNT, in this process. This comprehensive analysis served as a foundation of the molecular mechanisms underlying pNC migration, proliferation, and differentiation, providing potential clinical targets during corneal development and induction signals for corneal regeneration.

Browsing by Author "Warmflash, Aryeh"

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